MSACL 2023 Abstract

INTRODUCTION: Measurement of enzymatic activity in newborn dried blood spots (DBS) is the preferred first-tier method in newborn screening (NBS) for the mucopolysaccharidoses (MPSs). However, false positives are observed due mainly to the presence of pseudodeficiencies. Recent research has shown that second-tier measurement of glycosaminoglycan (GAG) biomarker levels in DBS for the MPSs can dramatically reduce the false positive rate in NBS. Additionally, these methods are useful tools in monitoring progression and treatment of MPSs and GM1 gangliosidosis, another Lysosomal Storage Disorder (LSD) which warrants GAG analysis.

OBJECTIVES: Two methods for measuring GAG biomarkers in 10 MPS conditions and GM1 gangliosidosis are directly compared in the analysis of DBS from newborns with positive diagnoses.

METHODS: MPS and GM1 gangliosidosis-diagnosed newborn DBS samples are analyzed against a reference range of healthy newborn DBS via two GAG mass spectrometry methods: (1) the internal disaccharide biomarker method, and (2) the endogenous biomarker method. The internal disaccharide method is the classical method for GAG analysis wherein GAG polymers in patient DBS are cleaved by bacterial lyases and hydrolases to yield many copies of a set of disaccharides from the repeating unit of GAG polymers. These disaccharides are then quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A second method, the endogenous biomarker method, analyzes the non-reducing end (NRE) of the GAG polymers without in vitro enzymatic digestion. Rather, endogenous NRE fragments generated by endohydrolases and exohydrolases in the human donor are derivatized and quantified directly by LC-MS/MS. A third NRE method, called the SensiPro method, involves enzymatic depolymerization of the GAG polymer, derivatization of the resulting glycans with aniline, and selective detection of the NRE derivative by LC-MS/MS. Due to similarities between the SensiPro method and the Internal Disaccharide method, in addition to complex sample workup requirements, the SensiPro method was only studied for MPS-I newborns.

RESULTS: The minimum differential factor (lowest GAG marker level in patient samples divided by highest level in a reference range of random newborns) was determined for both biomarker methods tested. The internal disaccharide minimum differentials for MPS-I and MPS-II were 2.5-fold and 3.0-fold, for MPS-IIIA and -IIIB were 3.7-fold and 5.4-fold, for MPS-IIIC and -VI were 9.3-fold and 2.1-fold, and for MPS-VII was 4.2-fold. The endogenous biomarker method minimum differentials for MPS-I and MPS-II were 16.0-fold and 3.8-fold, for MPS-IIIA and -IIIC were 4.6-fold and 3.2-fold, for MPS-IVA and-VI were 20.0-fold and 20.6-fold, and for MPS-VII and GM1 gangliosidosis were 10.0-fold and 3.0-fold, respectively. Non-newborn MPS-IIID DBS were tested for MPS-IIID against a reference range of healthy non-newborn DBS. The minimum differentials for these MPS-IIID DBS were 2.3-fold for the internal disaccharide method and 14.7-fold for the endogenous biomarker method.

CONCLUSION: Overall, the endogenous biomarker method outperforms the internal disaccharide method with regard to low variability in the reference range. While the internal disaccharide biomarkers give significant peak signals in healthy newborn DBS, the peak signals for endogenous biomarkers in healthy newborn DBS are either background noise or an order of magnitude lower than signals in patient DBS. Based on minimum differentials, the internal disaccharide method is the preferred method for MPS-IIIB and -IIIC but it is not able to discriminate MPS-IVA or GM1 gangliosidosis newborns from the reference range. The endogenous biomarker method is the preferred method for MPS-I, MPS-II, MPS-IIIA, -IIID, -IVA, -VI, -VII, and GM1 gangliosidosis and is generally the most useful method, with the exception that no endogenous marker was identified for MPS-IIIB DBS. MPS-IVB newborn DBS were not tested. This study supports the use of second-tier GAG analysis of newborn DBS, especially the endogenous biomarker method, as part of NBS to reduce the false positive rate.